An approach utilized in drug therapy is to employ delivery vehicles which exhibit pharmaceutically protective or specific targeting properties. Vehicles such as liposomes, immunoliposomes and lipoproteins are capable of binding lipophilic pharmaceuticals and facilitating their cellular uptake by endocytosis leading to delivery of the pharmaceutical to lysosomes of target cells.
Presently, drugs that are designed to be lipophilic and compatible with lipophilic carriers capable of delivering the drug to cells by endocytosis do not have characteristics that allow them to reach other cellular compartments. Accordingly, a drug having sites of action in cellular compartments other than in lysosomes cannot be effectively delivered by lipophilic carriers to target cells by endocytosis with the degree of selectivity that may be desired. A need exists for a pharmaceutical that undergoes intracellular redistribution throughout a target cell after the pharmaceutical is preferentially delivered to the lysosome of that cell.
Selective photocytotoxicity is the basis of photodynamic therapy (PDT). The PDT process requires photosensitizers (PS) that exhibit high selectivity and effective photoactivity at wavelengths suitable for significant tissue penetration. The first generation drug for PDT, hematoporphyrin derivative (HpD), shows considerable cross sensitization with non-target tissues and has absorption properties that require the use of light at wavelengths exhibiting only moderate tissue penetration. In an effort to improve upon the photoactive properties and the tissue selectivity of potentially useful PDT systems, attempts have been made to treat the elements of selectivity and photoactivity separately. Approaches have included the use of PS associated with liposomes and low-density lipoprotein (LDL). These avenues are premised, in part, on the initial use of the normal cellular mechanism of receptor-mediated endocytosis to direct the internalized material, through vesicular transport, to the lysosome. The endolysosomal compartments are acidic (.about.pH 4.8-6.5) and contain a host of hydrolytic enzymes. Lysosomal targeting of a PS has resulted in phototoxic responses in target cells under conditions wherein the PS is solely, or predominantly in the lysosomes. Damage to virtually all cellular compartments, however, has been implicated with photocytotoxicity in PDT. Accordingly, these results suggest that multi-site photodamage to a target cell, following delivery of the photosensitizer to the cell, may be an important mechanism that could facilitate cytotoxicity.
To design potentially useful PS applicable for lysosomal-targeted PDT that will allow a multisite response, it has been proposed to use pH-sensitive amphipathic PS, S. A. Chernomorsky, C. Wong and R. D. Poretz, "Pheophorbide a-induced photo-oxidation of cytochrome c: implication for photodynamic therapy", Photochem. Photobiol 55, 205-211 (1992). Such PS compounds exhibit a tendency to preferentially partition, depending upon the pH of the environment, into lipophilic membranes or into an aqueous milieu. Such compounds, when delivered to the acidic lysosome, are relatively hydrophobic and tend to diffuse into the organelle membrane. Contact with the neutral pH of the adjacent cytosol, however, will result in conversion of the photosensitizer to a more hydrophilic anionic species, allowing for it to diffuse into that compartment and partition throughout the lipophilic and aqueous compartments of the cell.
A concern with pH-sensitive amphipathic compounds that are wedded to a lipophilic carrier, such as liposomes or LDL, is the potential tendency of such substances to partition out of the lipophilic carrier into more hydrophilic compartments prior to delivery into the cell, R. Pottier, and J. C. Kennedy, "The possible role of ionic species in selective biodistribution of photochemotherapeutic agents toward neoplastic tissue," J. Photochem. Photobiol. B. Biol., 8, 1-16 (1990).
Acyloxyalkyl esters of hydrophilic drugs have been utilized to enhance intestinal uptake of orally delivered pro-drugs, with the resulting enzymatic de-esterification yielding the more hydrophilic drug, A. B. A. Jansen and T. J. Russell, "Some novel penicillin derivatives", J. Chem. Soc., 1965, 2127-2132 (1965); H. Ferres, "Pro-drugs of .beta.-lactam antibiotics," Chem. Ind., 435-440 (1980).
Similarly, Tsien has employed acetoxymethyl esters of fluorescent compounds to allow for the passive diffusion of the esters through the cell membrane resulting in the liberation of the anionic dye by the action of cytosolic esterases, R. Y. Tsien, "A non-disruptive technique for loading calcium buffers into cells, Nature, 290, 527-528 (1981).
Chlorin e.sub.6 and pheophorbide a are chlorins which are known to exhibit significant photocytotoxic activity, L. C. Bergstrom, I. Vucenik, S. Chernomorsky and R. D. Poretz, "Targeted photoactive immunoliposomes are cytotoxic to human bladder carcinoma cells," FASEB J., 5, A1558, (1991); J. D. Spikes, "Chlorins as photosensitizers in biology and medicine," J. Photochem. Photobiol. B. Biol., 6, 259-274 (1990); S. A. Chernomorsky, R. D. Poretz and A. B. Segelman, "The photodynamic effect of chlorophyll derivatives on murine myeloma cells in tissue culture," Photochem Photobiol., 39, 49S (1984); A. B. Segelman, I. K. Hagen, S. A. Chernomorsky, K. Weadock and G. H. Sigel, Jr., "Highly purified pheophorbide a as a photosensitizer in human bladder cancer in vitro," New Directions of Photodynamic Therapy, (Edited by D. C. Neckers), pp. 205-209, SPIE (1987); G. A. Kostenich, I. N. Zhuravkin, A. V. Furmanchuk and E. A. Zhavrid, "Photodynamic therapy with chlorin e.sub.6. A morphologic study of tumor damage efficiency in experiment," J. Photochem. Photobiol. B. Biol., 11, 307-318 (1991); G. A. Kostenich, I. N. Zhuravkin, A. V. Furmanchuk and E. A. Zhavrid, "Sensitivity of different rat rumour strains to photodynamic treatment with chlorin e.sub.6," J. Photochem. Photobiol. B. Biol., 17, 187-194 (1993).
The photocytotoxic effect of chlorin e.sub.6 is known to be greatly enhanced when it is delivered to intracellular compartments by specific carriers, A. R. Oseroff, D. Ohuoha, T. Hasan, J. C. Bommer and M. L. Yarmush "Antibody targeted photolysis: selective photodestruction of human T-cell leukemia cells using monoclonal antibody-chlorin e.sub.6 conjugates," Proc. Nat'l. Acad. Sci. USA, 83, 8744-8748 (1986); T. V. Akhlynina, P. V. Gulak, N. V. Serebryakova, A. A. Rosenkranz and A. S. Sobolev "Photodynamic action of concanavalin A-Chlorin e.sub.6 conjugate on human fibroblasts," Byull. Eksp. Biol. Med., 109, 150-152 (1990); J. Kopecek, N. L. Krinick, B. Rihova and K. Ulbrich "Targetable N-(2-hydroxypropyl) methacrylamide copolymer-chlorin e.sub.6 conjugates," Photodynamic Therapy: Mechanisms II, (Edited by T. J. Dogherty), pp. 144-152, SPIE, (1990); R. Bachor, C. R. Shea, S. J. Belmonte and T. Hasen "Free and conjugated chlorin e.sub.6 in the photodynamic therapy of human bladder carcinoma cells," J. Urol., 146, 1654-1658 (1991). R. Bachor, M. Scholz, C. R. Shea and T. Hasan, "Mechanism of photosensitization by microsphere-bound chlorin e.sub.6 in human bladder carcinoma cells," Cancer Res., 51, 4410-4414 (1991); A. S. Sobolev, T. V. Akhlynina, S. V. Yachmenev, A. A. Rosenkranz and E. S. Severin, "Internalizable insulin-BSA-chlorin e.sub.6 conjugate is a more effective photosensitizer than chlorin e.sub.6 alone," Biochem. Int., 26, 445-450 (1992). This enhancement phenomenon apparently is due to the potential trianionic charge of the PS at neutral pH values, thereby resulting in a membrane-impermeable form, R. Pottier and J. C. Kennedy, "The possible role of ionic species in selective biodistribution of photochemotherapeutic agents toward neoplastic tissue," J. Photochem. Photobiol. B. Biol., 8, 1-16 (1990).